荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验 替代方法的研究与应用

doi:10.3864/j.issn.0578-1752.2013.01.019

中国农业科学 ›› 2013, Vol. 46 ›› Issue (1): 162-169.doi: 10.3864/j.issn.0578-1752.2013.01.019

荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验替代方法的研究与应用

陈锴, 姚华伟, 王长江, 徐璐, 范学政, 赵启祖, 邹兴启, 朱元源, 赵燕, 杨光友, 王琴

1.四川农业大学动物医学院，四川雅安 625014
2.中国兽医药品监察所，北京 100081
3.新疆维吾尔自治区畜牧科学院，乌鲁木齐 830000

收稿日期:2012-10-17 出版日期:2013-01-01 发布日期:2012-12-24
通讯作者: 通信作者杨光友，Tel：0835-2882253；通信作者王琴，Tel：010-62103680，E-mail：wq551@vip.sina.com
作者简介:陈锴，E-mail：davis0591@gmail.com
基金资助:
“十一五”国家支撑计划课题（2006BAD06A18，2006BAD06A12）、新疆维吾尔自治区2012科技援疆项目（201291150）

Fluorescent Quantitative PCR as an Alternative Method for Efficacy Testing of Lapinized Hog Cholera Virus

CHEN Kai, YAO Hua-Wei, WANG Chang-Jiang, XU Lu, FAN Xue-Zheng, ZHAO Qi-Zu, ZOU Xing-Qi, ZHU Yuan-Yuan, ZHAO Yan, YANG Guang-You, WANG Qin

1.College of Veterinary Medicine, Sichuan Agricultural University, Ya’an 625014, Sichuan
2.China Institute of Veterinary Drug Control, Beijing 100081
3.Institute of Animal Science, Academy of Xinjiang Uygur Autonomous Region,Urumqi 830000

Received:2012-10-17 Online:2013-01-01 Published:2012-12-24

摘要/Abstract

摘要： 【目的】建立一种快速、敏感、特异的检测猪瘟兔化弱毒疫苗（HCLV）的荧光定量PCR方法(FQ-PCR)，为猪瘟兔化弱毒疫苗的效力检验提供一替代方法。【方法】在猪瘟兔化弱毒疫苗基因组3′非编码区设计一对针对猪瘟兔化弱毒株的特异性引物和一条MGB探针，并进行反应体系及反应条件优化，以及特异性、灵敏度、稳定性及符合性试验。将此方法用于部分批次疫苗厂疫苗半成品的定量检验，与兔热反应进行比较，寻求两方法的相关性。【结果】试验结果证实，CT值与模板之间呈现良好的相关性，相关系数为0.9998，扩增效率为101.14%；该方法仅扩增HCLV，不扩增HCLV以外的其它病原，显示良好的特异性；具有较高的灵敏度，能检测模板中4.35个cDNA拷贝量，比CSFV-RT-nPCR高1个数量级。将此方法应用于4个厂家17个批次的34份猪瘟兔化弱毒疫苗效力检验，两只兔子均无热反应（不合格）样品共11份，FQ-PCR CT值为21.15—27.30；两只兔子一只呈现定型热，一只无热反应的样品12份，CT值为17.47—23.70；两只兔子都呈现定型热或者一只呈现定型热一只呈现轻热反应（合格品）样品共11份，CT值为17.10—20.8。【结论】建立的HCLV-FQ-PCR方法能特异性的检测疫苗核酸含量，与家兔热反应测定结果存在良好的相关性，可以用于猪瘟疫苗半成品中HCLV定量检测。

关键词: 猪瘟兔化弱毒疫苗 , 效价检验 , 荧光定量PCR , TaqMan-MGB

Abstract: 【Objective】 A rapid, sensitive and specific one-step fluorescent quantitative PCR method as a substitute for rabbit fever testing for hog cholera lapinized virus (HCLV) vaccine efficacy was established. 【Method】 A pair of primers and a HCLV specific MGB probe were designed on the 3’UTR region of the HCLV genome for fluorescent quantitative PCR (FQ-PCR). The method was tested for specificity, sensitivity and conformity after optimization. 【Result】 The FQ-PCR sensitivity was 4.35 cDNA copies. The correlation coefficient between CT value and cDNA copies was 0.9998. Amplification efficiency was 101.14%. The FQ-PCR method was specific for HCLV and did not show amplifications for CSFV, BVDV, BDV, PRRSV, FMDV and other pathogens. A total of 34 samples from 17 batches of the four vaccine manufacturers were tested after serial dilutions using the rabbit fever test and FQ-PCR. Eleven samples were disqualified for lack of fever in two rabbits with the Ct values falling between 21.15 and 27.30 and viral content of 8.80×102copy/μL-6.52×104copy/μL. Twelve samples induced fever in one rabbit and no fever in the others with the Ct values between 17.47 and 23.70 and viral content of 1.10×104copy/μL-8.55×105copy/μL. The Ct values of 11 positive samples with both rabbits showing typical fever were from 17.10 to 20.81 with the viral content of 8.27×104copy/μL-1.11×106copy/μL.【Conclusion】The FQ-PCR kits established in this study is specific, sensitive and positively correlated with rabbit fever testing, and thus could be used for quantitative examination of semi-finished HCLV vaccine.

Key words: Lapinized hog cholera virus , efficacy testing , fluorescent quantitative PCR , TaqMan-MGB

陈锴, 姚华伟, 王长江, 徐璐, 范学政, 赵启祖, 邹兴启, 朱元源, 赵燕, 杨光友, 王琴. 荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验替代方法的研究与应用[J]. 中国农业科学, 2013, 46(1): 162-169.

CHEN Kai, YAO Hua-Wei, WANG Chang-Jiang, XU Lu, FAN Xue-Zheng, ZHAO Qi-Zu, ZOU Xing-Qi, ZHU Yuan-Yuan, ZHAO Yan, YANG Guang-You, WANG Qin. Fluorescent Quantitative PCR as an Alternative Method for Efficacy Testing of Lapinized Hog Cholera Virus[J]. Scientia Agricultura Sinica, 2013, 46(1): 162-169.

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荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验替代方法的研究与应用

Fluorescent Quantitative PCR as an Alternative Method for Efficacy Testing of Lapinized Hog Cholera Virus

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荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验 替代方法的研究与应用

Fluorescent Quantitative PCR as an Alternative Method for Efficacy Testing of Lapinized Hog Cholera Virus

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荧光定量PCR作为猪瘟兔化弱毒疫苗效价检验替代方法的研究与应用